Identification of post-transcriptionally regulated targets by TrIP-Chip/Seq

描述（申请人提供）：化疗耐药是癌症治疗失败的主要原因之一。转录后和翻译调控在化疗耐药性中起着重要的作用.目前，还没有可用的高通量方法来研究由来自少量细胞的RNA结合蛋白或非编码microRNA介导的转录后和翻译控制。我们在拟议项目中的主要目标是应用我们新开发的翻译免疫沉淀-微阵列分析（TrIP-Chip）（1）从少量结肠癌干细胞中发现由miR-215介导的转录后调节mRNA靶点。我们最近的研究表明，miR-215参与调节一些关键的抗癌靶点，例如胸苷酸合成酶和二氢叶酸还原酶。胸苷酸合成酶（TS）是一种叶酸依赖性酶，催化5，10-亚甲基四氢叶酸还原甲基化dUMP形成dTMP和二氢叶酸。由于TS催化的酶促反应提供了唯一的细胞内从头来源的胸苷酸，DNA生物合成的必要前体，TS已超过50年的癌症化疗的重要目标。二氢叶酸还原酶（DHFR）催化二氢叶酸还原为四氢叶酸的NADPH依赖性还原。该反应提供了单碳转移反应中的关键中间体。DHFR在叶酸稳态中起关键作用，并且是嘌呤、胸苷酸和某些氨基酸的从头合成所需的。因此，DHFR已成为癌症化疗的关键靶点。通过基于多维蛋白质鉴定技术（MudPIT）的灵敏的高通量鸟枪蛋白质组学分析，将在蛋白质水平上验证由miR-215转录后调节的mRNA。我们将进一步开发一种TrIP-Seq方法，以提高在单细胞水平上检测稀有转录本的覆盖率和灵敏度。提出了三个具体目标：具体目标1。使用TrIP-Chip方法鉴定miR-215的转录后和转录调控mRNA靶点。具体目标2。通过高通量蛋白质组学分析确定miR-215介导的靶点。具体目标3。我们将通过整合下一代基于测序的表达分析（TrIP-Seq）来进一步开发TrIP-Chip方法，以增加少量细胞的覆盖率和检测灵敏度。 公共卫生相关性：目前，缺乏用少量细胞研究转录后和翻译调控的高通量方法。我们已经开发了一种新的翻译免疫沉淀阵列基因表达分析（TrIP芯片）技术，使我们能够调查转录后和翻译调控基因从少至500个细胞。我们相信，随着下一代深度测序平台（例如Illumina Instrument）的动态范围的改善，我们可以潜在地分析来自单个细胞的翻译控制。该项目将开发和应用这种新方法，以发现miR-215介导的靶点和在结肠癌和结肠癌干细胞中的功能意义。这项技术不仅在癌症方面有广泛的应用，而且在其他人类疾病方面也有广泛的应用。

DESCRIPTION (provided by applicant): Resistance to chemotherapy is one of the major reasons for the failure of cancer treatment. Post- transcriptional and translational control plays an important role in chemoresistance. Currently there is no available high throughput approach to investigate post-transcriptional and translational control mediated by RNA binding proteins or non-coding microRNAs from a small number of cells. Our primary objective in the proposed project is to apply our newly developed Translational Immunoprecipitation-Microarray Analysis (TrIP-Chip) (1) to discover post-transcriptionally regulated mRNA targets mediated by miR-215 from a small number of colon cancer stem cells. Our recent studies revealed that miR-215 is involved in regulating some key anticancer targets, e.g. thymidylate synthase and dihydrofolate reductase. Thymidylate synthase (TS) is a folate-dependent enzyme that catalyzes the reductive methylation of dUMP by 5,10-methylenetetrahydrofolate to form dTMP and dihydrofolate. Because the TS-catalyzed enzymatic reaction provides the sole intracellular de novo source of thymidylate, an essential precursor for DNA biosynthesis, TS has been an important target for cancer chemotherapy for over 50 years. The enzyme dihydrofolate reductase (DHFR) catalyzes the NADPH-dependent reduction of dihydrofolate to tetrahydrofolate. This reaction provides the key intermediate in one-carbon transfer reactions. DHFR plays a critical role in folate homeostasis, and is required for the de novo synthesis of purines, thymidylate and certain amino acids. Therefore, DHFR has served as a critical target in cancer chemotherapy. The mRNAs regulated post-transcriptionally by miR-215 will be validated at the protein level by a sensitive, high throughput shotgun proteomic analysis based on multi-dimensional protein identification technology (MudPIT). We will further develop a TrIP-Seq approach to increase both the coverage and sensitivity to detecting rare transcripts potentially at the single cell level. Three specific aims are proposed: Specific Aim 1. Identify post-transcriptionally and translationally regulated mRNA targets of miR-215 using TrIP-Chip approach. Specific Aim 2. Validate miR-215 mediated targets by high throughput proteomic analysis. Specific Aim 3. We will further develop the TrIP-Chip approach by integrating next generation sequencing based expression analysis (TrIP-Seq) to increase coverage and detection sensitivity from a small number of cells. PUBLIC HEALTH RELEVANCE: Currently, there is a lack of high throughput approach to study post- transcriptional and translational control with a small number of cells. We have developed a novel Translational Immunoprecipitation-Array Gene expression analysis (TrIP-Chip) technology that allow us to investigating post-transcriptional and translational regulated genes from as few as 500 cells. We believe with the improvement of the dynamic range of the next generation deep sequencing platform (e.g. Illumina Instrument), we can potentially analyze translational control from a single cell. The proposed project will develop and apply this new approach to discover miR-215 mediated targets and functional significance in colon cancer and colon cancer stem like cells. The technology will have a broad application not only in cancer but other human diseases as well.